The rational design and implementation of the next generation of therapies and vaccines against Mycobacterium tuberculosis will require a thorough understanding of the mechanisms of antibiotic resistance and bacterial pathogenesis as they apply to human infections. In this proposal, population-based genetic studies of human specimens will be used to determine the clinical consequences of mutations in genes associated with bacterial antibiotic resistance and virulence. Previous population based genetic studies of M. tuberculosis have been limited in scope due to the difficulty and expense of large scale DNA sequencing or DNA chip analysis. The applicants have developed molecular beacon PCR assays which are expected to allow them to rapidly and accurately screen large numbers of samples for specific genetic mutations with the accuracy of a single base pair. They will screen for the presence of larger insertions and deletions in DNA sequence using a modified slot blot cross- hybridization approach. They propose to use novel transducing phage techniques to induce homologous recombination and gene substitution in M. tuberculosis. These techniques and more established methods will be used to study clinical M. tuberculosis isolates acquired by three large scale epidemiological studies underway in Arkansas, San Francisco, and Orizaba, Mexico. DNA sequence analysis will be combined with classical and molecular epidemiology to study the mutational events that lead to resistance to isoniazid (INH), to determine the distribution of specific mutations in susceptible and resistant isolates, and to discover other mechanisms of isoniazid resistance that develop in clinical strains. Transducing phage assays will be used to uncover and characterize additional resistance mutations. A similar approach will be applied to investigate infectivity and virulence in clinical M. tuberculosis strains. The complete genomic sequence differences between the laboratory strain H37Rv, and a highly virulent clinical strain CSU093, have recently become known. Studies of these sequence variations in clinical strains will be used to determine the associations between specific mutations and the phenotypes of infectivity and virulence.